Gram-positive bacteria are collectively classified as having a single lipid bilayer plasma membrane. Gram positive bacteria include a multitude of bacilliform and cocciform bacterial genera, among which Bifidobacteria and a group of genera collectively known as lactic acid bacteria (LAB). LAB comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics. These bacteria, usually found in (decomposing) plants and dairy products, produce lactic acid as the major metabolic end-product of carbohydrate fermentation. This trait has, throughout history, linked LAB with food fermentations, as acidification inhibits the growth of spoilage agents. A prototype LAB Lactococcus lactis is a mesophilic and microaerophilic fermenting lactic acid bacterium. While the bacterium is extensively used in food fermentations, especially in the dairy industry, there is an increasing interest for its use in medicaments and nutraceuticals, as medication to treat infections in bodily cavities such as vaginal infections, or as carrier for the delivery of biological active molecules. In all those cases, there is a need for highly viable starter cultures, or pharmaceutical or nutraceutical formulations comprising a high proportion of viable bacteria. L. lactis, however, tends to lose viability during storage, or during processing (for a.o the production of a dry powder formula, tablet formation, . . . ). The drop in viability is even more pronounced when the bacterium after lyophilisation is submitted to additional stress such as high acidity or the presence of bile salts.
Several methods have been proposed to overcome this problem. The use of trehalose is of particular interest. Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a non-reducing disaccharide that occurs in a large variety of organisms, ranging from bacteria to invertebrate animals. Trehalose, sometimes in combination with dextran, is often used as and externally added cryopreservant. Externally added trehalose functions as a saccharide matrix (Conrad et al., 2000,), and exerts it protective effect especially during freeze drying, where it acts as a glass former. Moreover, trehalose is well recognized as stress metabolite, and it has been extensively studied in fungi, especially in Saccharomyces cerevisiae. High concentrations of internal trehalose do improve the storage capacity and result in a higher viability upon cryopreservation. However, it is important to note that externally added trehalose rarely leads to internal trehalose accumulation in micro-organisms, either because it is not taken up, or it is metabolized rapidly after uptake.
Termont et al. (Appl Environ Microbiol 72:7694; 2006) reported that de-novo synthesized trehalose, through plasmid driven overexpression of otsA (trehalose-6-phosphate synthase) and otsB (trehalose-6-phosphate phosphatase) accumulates intracellularly in L. lactis. Intracellular trehalose accumulation but not exogenously added trehalose protects L. lactis from bile lysis and cell death through freeze-drying. As L. lactis is extremely sensitive, protection to bile lysis can be used as a superb functional assay of intracellular trehalose accumulation.
Andersson et al. (J Biol Chem 276:42707; 2001) have described a novel pathway for trehalose utilization in L. lactis. This pathway employs the activity of trehalose-6-phosphate phosphorylase (trePP), converting trehalose-6-phosphate to β-glucose 1-phosphate and glucose 6-phosphate. They describe insertional inactivation of trePP in L. lactis, resulting in loss of capacity to grow on trehalose.
For the intracellular accumulation of trehalose, Carvalho et al. (Appl Environ Microbiol 77:4189; 2011) describe a method that makes use of plasmid driven overexpression of L. lactis trePP and β-phosphoglucomutase (pgmB). As indicated by these authors, given that the bacteria lack trehalose 6-phosphate phosphatase, the respective gene, otsB, from food-grade organism P. freudenreichii was used to provide the required activity. The resulting cells showed improved resistance to cold shock, heat shock and acidity. However, the authors indicated that at least 67% of the trehalose produced was found in the growth medium. Hence the produced trehalose appears not to be efficiently retained or accumulated intracellularly.
Although these processes certainly lead to an improvement of the storage, there is a further need of methods that can lead to an improved storage of gram positive bacteria, such as LAB or Bifidobacteria, not only in those cases where the bacterium is used for the delivery of biological active compounds in medical applications, but also when the bacterium is used in the food industry, such as the dairy industry.
Lowes et al. 2006 (Oral Microbiol Immunol. 21(1): 21-7) discloses certain mutants of Streptococcus mutans bacterium, which he denotes as PTS system IIC component (PtcC) mutants. S. mutans as studied by Lowes is a pathogen causing dental caries, and Lowes is ultimately concerned with investigating genomic variability of S. mutans in the context of its pathogenicity. Utilisation of beta-glucoside carbohydrate sources may play a role in pathogenicity and survival of S. mutans, and PtcC is investigated from this perspective. Lowes does not suggest any role of PtcC in internal accumulation of trehalose, nor in improving stress resistance of bacteria. Notably, Lowes et al. 2006 studies metabolism of beta-glucosides, whereas trehalose is an alpha-glucoside. Lowes does not concern PtcC mutants of non-pathogenic bacteria or any utility of such mutants.